Lignins are universal components in plants that form cross-links with carbohydrates, such as hemicelluloses in the cell wall. Lignin and cellulose are the two predominant components of the plant cell wall. The plant cell wall provides a natural barrier against the extracellular environment. Many studies have demonstrated that one of the responses of plants to biotic stresses (e.g., pathogenic infection) or abiotic stresses (e.g., drought, mechanical stress, etc.) consists of reinforcement of the plant cell wall, in particular by increasing the lignin content in the plant cell wall. Many agronomical or industrial applications concern desired plant products (e.g., products used in paper production, silage production, and the production of energy, for example, in the form of biofuels), the yields of which are directly linked to the content and/or composition of lignin in the plant cell wall.
Lignin polymers limit the digestibility of the fiber in the corn plant. Lignin polymers lower fiber digestion in ruminants, and the degree of lignifications may be inversely proportional to forage crop digestibility. Cherney et al. (1991) Adv. Agron. 46:157-98. Modulation of lignin content and composition may be desirable to increase the digestibility of forage. Lignin content modulation may also be desirable, e.g., to reinforce plant walls, and thereby improve resistance to stresses; or conversely to weaken the plant wall in order to facilitate the extraction of cellulose or other chemical compounds. Baucher et al. (1998) Plant Mol. Biol. 39:437-47.
It is, however, difficult to know how to modify the lignin biosynthesis pathway, and to predict what the consequences of modifications will be. This is at least in part because the lignin biosynthesis pathway is a complex pathway involving a large number of enzymatic reactions. See, e.g., Dixon et al. (2001) Phytochemistry 57(7):1069-84. Possible mechanisms by which the pathway may be altered physiologically, for example, to compensate for a change introduced by a modification in the pathway, are not known.
Lignin is an insoluble polymer of 3 monomers of alcohols or monolignols: p-coumaryl alcohol (H subunits), coniferyl alcohol (G subunits), and sinapyl alcohol (S subunits), that are derived from the phenylpropanoid pathway. Neish (1968) Constitution and Biosynthesis of Lignin, eds. New York, Springer Verlag 1-43. Each type of subunit can form a variety of bonds with others, and thereby constitute lignin. Other bonds may also be established with other parietal compounds (e.g., polysaccharides and proteins) so as to form a complex three-dimensional network.
Steps in the complex lignin production pathway include hydroxylation, O-methylation of aromatic rings, and conversion of a carboxyl side chain to an alcohol function. The current hypothesis for the monolignol biosynthesis pathway includes successive hydroxylation and O-methylation reactions at various levels of oxidation of the side chains in a metabolic network, thereby resulting in the formation of S and G subunits. The enzymes of the network include caffeic acid 3-O-methyltransferase (COMT); hydroxyxinamate coenzyme A ligases (4CL); cytochrome P450-dependent ferulate 5-hydroxylases (F5Hs); and several isoforms of cinnamoyl CoA reductase (CCR) and of cinnamyl alcohol dehydrogenase (CAD).
For several years, attempts have been made to modify the lignin content and composition of plants by over-expressing or under-expressing one or more genes of the lignin biosynthesis pathway. Anterola and Lewis (2002) Phytochemistry 61:221-94. Though various strategies have been imagined, the over-expression or under-expression of one or more enzymes in the lignin biosynthesis pathway does not always give reliable and predictable results.
Another strategy consists of using, in selection schemes, mutants of a targeted gene in the lignin biosynthesis pathway. Plants containing a brown midrib (bmr) mutation exhibit altered lignin composition and digestibility. In corn, at least four independent brown midrib mutations have been identified. Kuc et al. (1968) Phytochemistry 7:1435-6. These mutations, termed “bm1, bm2, bm3, and bm4,” all exhibit decreased lignin content when compared to control corn. Brown midrib corn plants are characterized by a brown pigmentation in the leaf midrib at the V4 to V6 stage and a light brown coloration of the pith after tasselling. One characterized bmr mutation is an insertion mutation in the COMT enzyme (bm3).
Mature bm1 maize plants have a lignin content that is reduced by 10-20%, a slight decrease in ferulic acid esters, and a substantially reduced content (˜40%) of p-coumaric esters and ferulic acid esters. Provan et al. (1997) J. Agric. Food 73:133-42; Barriére et al. (2004) Comptes Rendus Biologie 327:847-60. The frequency of p-hydroxyphenyl, guaiacyl, and syringyl thioacidolysis monomers is similar in bm1 and wild-type plants, showing that the bm1 mutation does not specifically affect a single type of lignin subunit. Guillaumie et al. (2007) Planta 226(1):235-50. Lignins of bm1 plants do appear to be substantially enriched in carbon-carbon inter-subunit linkages (Halpin et al. (1998) Plant J. 14(5):545-53; Barriére et al. (2004), supra), and bm1 lignins have substantial incorporation of coniferaldehyde and, to a lesser extent, of sinapaldehyde. Kim et al. (2002) J. Biol. Chem. 277:47412-9.
Agriculturally important uses of corn (maize) include silage. Silage is fermented, high-moisture fodder that can be fed to ruminants. It is fermented and stored in a process called ensilage or silaging, and is usually made from corn or other grass crops, including sorghum or other cereals, using the entire green plant. Bulk silage is commonly fed to dairy cattle, while baled silage tends to be used for beef cattle, sheep, and horses. Since silage goes through a fermentation process, energy is used by fermentative bacteria to produce volatile fatty acids, such as acetate, propionate, lactate, and butyrate, which preserve the forage. The result is that the silage is lower in energy than the original forage, since the fermentative bacteria use some of the carbohydrates to produce the volatile fatty acids. Corn silage is a popular forage for ruminant animals because it is high in energy and digestibility and is easily adapted to mechanization from the stand-crop to time of feeding. Corn silage generally is slightly brown to dark green in color, and has a light, pleasant smell.
The reduced lignin in brown midrib corn (bmr corn) results in silage with fiber that is more digestible than normal corn and exhibits an improved rate of biofuel conversion. Feeding bmr corn silage to lactating dairy cows has been shown to increase dry matter intake (DMI) and milk yield. Grant et al. (1995) J. Dairy Sci. 78:1970-80; Oba and Allen (2000) J. Dairy Sci. 83:1333-41; Oba and Allen (1999) J. Dairy Sci. 82:135-42. However, bmr corn silage reduced average daily gain and feed efficiency (G:F) in beef cows, compared to corn silage from a conventional corn variety. Tjardes et al. (2000) J. Anim. Sci. 78:2957-65. Brown midrib hybrid corn lines are also frequently found to be low yielding. Brown midrib hybrid corn has also typically been associated with forage lodging and lack of standability.